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EP-4735893-A1 - TARGETED PROTEOMICS FOR MONITORING AUTOPHAGY

EP4735893A1EP 4735893 A1EP4735893 A1EP 4735893A1EP-4735893-A1

Abstract

The invention relates to a peptide mix comprising peptides for use in monitoring autophagy, the peptides being derived from proteins involved in autophagy.

Inventors

  • STUMPE, MICHAEL
  • LEYTENS, Alexandre
  • DENGJEL, Jörn

Assignees

  • University Of Fribourg

Dates

Publication Date
20260506
Application Date
20240628

Claims (15)

  1. 1. A method for monitoring autophagy selectivity, the method comprising probing a sample via mass spectrometry for the amount of at least two different selective autophagy receptor (SAR) proteins using a peptide mix as peptide standard, wherein the peptide mix comprises peptides derived from the at least two different SAR proteins.
  2. 2. The method according to claim 1, wherein the at least two different SAR proteins are specific for at least two different autophagy types selected from aggrephagy, mitophagy, pexophagy, lysophagy, zymophagy, ER-/reticulo-phagy, ferritinophagy, glycophagy, xenophagy, ribophagy, midbody autophagy, golgiphagy, nucleophagy and lipophagy.
  3. 3. The method according to claim 1 or 2, wherein the SAR proteins are selected from the group consisting of SQSTM1, NBR1, Optineurin, CALCOCO2, TAX1BP1 , Alfy, CCPG1, FAM134A, FAM134B, FAM134C, ATL3, BCL2L13, CDK5RAP3, GOLPH3, LMNB1 , SEC62, SPART, TEX264, YIPF3, YIPF4, RTN3, NIX/BNIP3L, TOLLIP, TNIP1, FKBP8, BNIP3, PHB2, NCOA4, NUFIP1 , STBD1, BNIP1 , PHBP2 and PNPLA2.
  4. 4. The method according to any of claims 1 to 3, wherein the peptide mix comprises one or more peptides having SEQ ID NO: 1, 2, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 78, 79, 80, 81, 82, 92, 93, 115, 116, 117, 118, 119, 120, 121, 122, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 148, 149, 150, 151 , 152, 153, 154, 168, 169, 174, 175, 176, 177, 190, 191 , 192, 193, 194, 206, 207, 208.
  5. 5. The method according to any of claims 1 to 4, wherein the peptide mix further contains peptides derived from proteins that are involved in autophagy regulation, in autophagosome initiation, in membrane nucleation, in membrane expansion, in pore closure and/or in fusion of the autophagosome with lysosomes.
  6. 6. The method according to any of claims 1 to 5, wherein the proteins involved in autophagy regulation are selected from the group consisting of ribosomal S6 kinase (RPS6KB1) and TFEB.
  7. 7. The method according to any of claims 1 to 6, wherein the proteins involved in autophagosome initiation are selected from the group consisting of LILK1 , LILK2, RB1CC1/FIP200 and ATG13.
  8. 8. The method according to any of claims 1 to 7, wherein the proteins involved in membrane nucleation are selected from the group consisting of ATG9A, AMBRA1 , and WBP2.
  9. 9. The method according to any of claims 1 to 8, wherein the proteins involved in membrane expansion are selected from the group consisting of ATG2A, WIPI1, WIPI2, WIPI3, WIPI4, ATG3, ATG7, ATG12, ATG5 and ATG4A.
  10. 10. The method according to any of claims 1 to 9, wherein the proteins involved in pore closure and fusion with lysosomes are GABARAPL1 or GABARAPL2.
  11. 11. The method according to any of claims 1 to 10, wherein the peptide mix comprises at least 7 peptides selected from the group consisting of peptides having SEQ ID NO: 1 to 213.
  12. 12. The method according to any of claims 1 to 11, wherein the peptide mix comprises at least one peptide derived from a protein involved in autophagy regulation, at least one peptide derived from a protein involved in autophagosome initiation, at least one peptide derived from a protein involved in membrane nucleation, at least one peptide derived from a protein involved in membrane expansion, at least one peptide derived from a protein involved in pore closure, at least one peptide derived from a protein involved in fusion with lysosomes and at least one peptide derived from a selective autophagy receptors (SAR) protein.
  13. 13. A mass spectrometry peptide standard comprising a peptide mix, wherein the peptide mix comprises peptides derived from at least two different SAR proteins, for use in a method according to any of claims 1 to 12.
  14. 14. The peptide standard according to claim 13, wherein the peptides are isotopically labelled heavy peptides.
  15. 15. The peptide standard according to claim 13 or 14, wherein the peptide mix comprises at least 7 peptides selected from the group consisting of peptides having SEQ ID NO: 1 to 213.

Description

Targeted proteomics for monitoring autophagy Field of the invention The invention relates to peptide mixes for use in mass spectrometry. Technical background Eukaryotic cells maintain homeostasis and remove damaged or superfluous cellular components through a conserved degradation pathway called macroautophagy (hereafter: autophagy). This highly conserved catabolic pathway occurs at basal levels but is enhanced by a variety of stress signals, such as nutrient starvation or organelle damage. The process starts with the de-novo formation of a double-membrane organelle called autophagosome from membranes being recruited from various sources, the endoplasmic reticulum (ER) likely being principle donor. Autophagosome biogenesis and turnover is a directional process and can be divided into five principle stages. In phase 1 “autophagosome initiation” kinase complexes are activated that start the process. In phase 2 “membrane nucleation” initial membrane sources are recruited to the phagophore assembly site, the principal site of autophagosome biogenesis. In phase 3 “membrane expansion” and phase 4 “pore closure”, the growing cup-shaped membrane engulfs parts of the cytoplasm and closes up to form a double membrane vesicle. The autophagosome can fuse with other vesicles from endocytic pathways forming an amphisome and finishes in phase 5 by “fusing with the lysosome/vacuole”. Lysosomal fusion exposes autophagosomal cargo including the inner membrane to acidic hydrolases and enables the degradation of its content. The generated building blocks are recycled and transported back to the cytosol by lysosomal permeases to generate energy or fuel anabolic pathways. While basal autophagy is often considered a “bulk process”, i.e., a non-selective process recycling cellular components in a random manner, autophagy can also be selective. Well studied cases of selective autophagy include the targeted removal of damaged organelles such as the mitochondrion or ER (termed mitophagy and reticulophagy/ER-phagy, respectively) as well as of protein aggregates (aggrephagy). This particular selectivity is enabled by a set of proteins called selective autophagy receptors (SARs), bridging the autophagy cargo to proteins of the ATG8 family, which are lipidated proteins coating the autophagosomal membrane and functioning as docking sites. Numerous methods and protocols have been described for the study of protein turnover by autophagy, several of them relying on immunodetection or fluorescent microscopy of single proteins, which are often ectopically expressed as tagged variants facilitating downstream analyses. While these methods are well established, they often require a significant protein amount or protein tagging, making them unsuitable for some sample types, e.g., primary cells being difficult to transfect, and difficult to adapt for large-scale screening approaches. Additionally, some of these methods infer the activity of the whole pathway based on the quantification of single markers. Whereas this might be relevant for the analysis of basal, i.e., non-selective, autophagy flux, such approaches likely fail to capture the complexity of stress-induced autophagy in which different cargoes might be degraded at different rates and through different pathways. To address the complexity of autophagy regulation and activity in an unbiased manner, approaches monitoring the activity of multiple genes by RNA-seq have been developed that are amendable for higher sample throughput and might also be used in prognostic/diagnostic settings. However, to infer autophagy activity based on changes in gene transcription biological samples have to be treated for several hours, commonly 2-8 hours, which is in contrast to the rapid cellular response to stress conditions. Autophagy signaling is activated within minutes, initial autophagosome biogenesis does not require de novo protein synthesis, and in mammalian cells autophagosomes and autophagydependent protein degradation can be detected as early as 30 min post stimulus. Objective problem to be solved To address this gap in analytical approaches, an assay is required that is able to detect changes in protein abundance, which is amenable to high throughput and which monitors abundance changes of multiple endogenous proteins reflecting the complexity and multitude of autophagy subtypes. Summary of the invention In one aspect, the invention relates to a method for monitoring autophagy selectivity, the method comprising probing a sample via mass spectrometry for the amount of at least two different selective autophagy receptor (SAR) proteins using a peptide mix as peptide standard, wherein the peptide mix comprises peptides derived from the at least two different SAR proteins. In a second aspect, the invention relates to a mass spectrometry peptide standard comprising a peptide mix, wherein the peptide mix comprises peptides derived from at least two different SAR proteins, for use in a method accordin